Chitosan-based nitric oxide donor compositions

ABSTRACT

A chitosan-based polymeric nitric oxide donor composition comprising a modified chitosan polymer and a nitric oxide [N 2 O 2 ] dimer, wherein the nitric oxide [N 2 O 2 ] dimer is bonded directly to the backbone of the modified chitosan polymer without further binding through a nucleophile residue or moiety. The chitosan-based polymeric nitric oxide donor composition is capable of site specific delivery and controlled release of nitric oxide under physiological conditions. The chitosan-based polymeric nitric oxide donor composition further provides a carrier having medically beneficial properties. A method is further included for preparing a chitosan-based polymeric nitric oxide donor composition comprising reacting a nitric oxide dimer (80-100 p.s.i.) with a modified chitosan polymer in the presence of sodium methoxide at room temperature. The chitosan-based polymeric nitric oxide composition can be incorporated into dry powder inhalers, wound dressings, implants, injectables, condoms, wound dressings and prosthesis coatings for use in a variety of medical applications in which an effective dosage of nitric oxide is indicated as a preferred method of treatment.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 09/199,732,filed Nov. 25, 1998. Now U.S. Pat. No. 6,261,594.

TECHNICAL FIELD OF THE INVENTION

The present invention is directed to a polymeric composition that iscapable of releasing nitric oxide (NO). The present invention is moreparticularly directed to a chitosan-based polymeric nitric oxide donorcomposition comprising a modified chitosan polymer and a nitric oxide[N₂O₂]⁻dimer, which provides site specific delivery and controlledrelease of nitric oxide under physiological conditions; pharmacologicalcompositions comprising the polymeric composition; and the use of thepolymeric composition to treat biological disorders for which aneffective dosage of exogenous nitric oxide is indicated as a preferredmethod of treatment.

BACKGROUND OF THE INVENTION

The chemical versatility of the nitric oxide (NO) molecule allows it toparticipate in a variety of physiologic processes. Nitric oxide (NO)reacts in biological systems with molecular oxygen (O₂), superoxide (O₂⁻) and transition metals to form high nitrogen oxides (NO_(x)),peroxynitrite (OONO⁻) and metal-nitrosyl adducts which have varioustoxicities and biological activities.

The role of the nitric oxide (NO) molecule has been implicated in manyphysiologic processes, such as regulation of pulmonary hypertension viaendothelium derived relaxing factor (EDRF)-induced vascular relaxation,central and peripheral neurotransmission, modulation of intestinalmotility, regulation of male erectile function, macrophage-inducedcytostasis and cytotoxicity, platelet inhibition, and non specificimmune response.

Nitric oxide synthases (NOS) catalyze the oxidation of the substrateL-arginine into L-citrulline and nitric oxide (NO) in a variety ofmammalian cell populations, such as pulmonary mucosa, submucosa, muscle,nerve and endothelium, reflecting the diverse biological activities ofnitric oxide (NO).

Investigations have been conducted which pharmacologically manipulatethe tissue level of nitric oxide (NO) by exogenous delivery of nitricoxide (NO) through inhalation of nitric oxide (NO) gas into the lungsvia an endotracheal tube during mechanical ventilation. Althoughinhalation of nitric oxide gas has been useful in the treatment of suchconditions as pulmonary hypertension, there are several disadvantagesand limitations with this particular mode of therapy. Nitric oxide (NO)inhalation therapy requires large gas tanks, expensive monitoringequipment, and a highly trained and skilled technician to operate thetanks and equipment, to deliver a therapeutically effective amount ofnitric oxide (NO) gas to a patient. Due to the volatility, hazardous andpoisonous properties of nitric oxide (NO) gas, safe delivery ofexogenous nitric oxide (NO) has been difficult to achieve.

In response to the great need in the art of nitric oxide (NO) therapy todevelop a more convenient, less expensive and safer method to deliverexogenous nitric oxide (NO) to patients, numerous donor compounds havebeen developed to administer an effective dosage of exogenous nitricoxide (NO) in biological systems under physiological conditions.

A number of compounds have been developed which are capable of binding,delivering and releasing nitric oxide (NO) in physiological conditionsupon being metabolized, including nitrovasodilators such as glyceryltrinitrite and sodium nitroprusside. (Ignarro et al., J. Pharmacol. Exp.Ther., 218, 739-749 (1981); Ignarro, Annu. Rev. Pharmacol. Toxicol., 30,535-560 (1990); Kruszyna et al., Toxicol. Appl. Pharmacol., 91, 429-438(1987); Wilcox et al., Chem. Res. Toxicol., 3, 71-76 (1990)). Althoughcompounds such as glyceryl trinitrite and sodium nitroprusside arerelatively stable under physiological conditions, a tolerance toglyceryl trinitrite via the exhaustion of the relevant enzyme/cofactorsystem is sometimes experienced in some applications, thus its use as anitric oxide donor is limited. (Ignarro et al., Annu. Rev. Pharmacol.Toxicol., 25, 171-191 (1985); Kuhn et al., J. Cardiovasc. Pharmaol., 14(Suppl. 11), S47-S54 (1989)). Furthermore, toxicity can develop fromprolonged administration of sodium nitroprusside due to the metabolicproduction of cyanide. (Smith et al., “A Potpourri of BiologicallyReactive Intermediates” in Biological Reactive Intermediates IV.Molecular and Cellular Effects and Their Impact on Human Health (Witmeret al., eds.), Advances in Experimental Medicine and Biology Volume 283(Plenum Press: New York, 1991), pp. 365-369).

A number of biodegradable compounds, such as primary, secondary, andpolyamines, have been developed which are capable of delivering nitricoxide (NO), and releasing nitric oxide (NO) in physiologic conditionsupon being metabolized. Keefer et al, U.S. Pat. No. 4,954,526 disclose amethod of treating cardiovascular disorders with an effective amount ofa stabilized complex formed from nitric oxide and primary amines andesters, ethers, of the formula [R—N(H)N(NO)—]_(y)X.

Further compounds have been developed that comprise diazenium diolates(NONOates) bound to polymers. NONOates are complexes of nitric oxide(NO) and nucleophiles (X⁻) in which a nitric oxide (NO) dimer is boundto the nucleophilic residue via a nitrogen atom, thus forming a NONOatehaving the following chemical formula:

Wherein R and R′ are the same or different, and constitute any of anumber of known organic moieties.

Traditionally, the synthesis of NONOates was accomplished by dissolvinga nucleophile in an organic solvent and exposing the solution to a fewatmospheres of NO gas for a period of a few days. A nucleophile, as theterm is used above, is defined as an ion or molecule that donates a pairof electrons to an atomic nucleus to form a covalent bond. (Hawley'sCondensed Chemical Dictionary, Twelfth Edition). Useful nucleophiles forthe synthesis of NONOates have traditionally included primary, secondaryor polyamines. When a polyamine, such as spermine is used as thenucleophile, zwitterions are formed. Upon exposure of the nucleophilesolution to NO gas, the NO dimer behaves as an electron pair acceptor,thus forming a covalent bond with the electron pair-donatingnucleophile. This reaction results in a nitric oxide/nucleophilecomplex. The nucleophile moiety of the nitric oxide/nucleophile adductcan be further be bound to a polymer, such as a polysaccharide, suchthat the nucleophile moiety of the nitric oxide/nucleophile complexforms part of the polymer itself. NONOates are useful as nitric oxidedonors in biological systems due to their ability to spontaneouslydisassociate under physiological conditions to regenerate the freenucleophile and molecular nitric oxide (NO).

Keefer et al, U.S. Pat. Nos. 5,250,550 and 5,155,137 disclose complexesof nitric oxide (NO) and polyamines, such as spermine and spermadine,useful in treating cardiovascular disorders, such as pulmonaryhypertension, and which release nitric oxide (NO) under physiologicalconditions in a sustained and controlled manner. These complexes aremade into pharmaceutical compositions by combination with medicallyacceptable carriers or diluents. Specifically, these compounds can beprepared into injectables by dissolving, suspending or emulsifying themin an aqueous or non-aqueous solvent.

Keefer et al, U.S. Pat. No. 5,212,204 discloses a method for loweringblood pressure using an antihypertensive composition comprising the N₂O₂⁻ functional group, an inorganic or organic moiety and apharmaceutically acceptable cation. The organic or inorganic moiety isany moiety that will form the anithypertensive composition and releasenitric oxide (NO) under physiological conditions upon decomposition.

Keefer et al, U.S. Pat. Nos. 5,208,233 and 5,039,705 disclose a methodof treating cardiovascular disorders that will respond to a decrease inblood pressure, such as chronic hypertension, acute congestive heartfailure, angina, acute myocardial infarction, left ventricular failure,cerebrovascular insufficiency and intracranial hemorrhage, by using anantihypertensive composition of secondary amines and nitric oxideadducts.

Keefer et al, U.S. Pat. No. 5,366,997 disclose a cardiovascularly activecomposition possessing antihypertensive properties comprising oxygensubstituted derivatives of nucleophile-nitric oxide adducts as nitricoxide donor prodrugs.

Christodoulou et al, U.S. Pat. No. 5,389,675 disclose mixed ligand metalcomplexes of nitric oxide-nucleophile adducts which are capable ofreleasing nitric oxide (NO), and are useful as cardiovascular agents.The nitric oxide-nucleophile complex ligand are coordinated via theoxygen donor atoms of the bidentate N₂O₂ ⁻ functionality to metalcenters, which are further bound to one or more additional ligands.

Smith et al, U.S. Pat. No. 5,691,423 disclose a polymeric compositioncapable of releasing nitric oxide (NO) in which the nitric oxidereleasing functional group [N₂O₂]⁻ is bound to the polysaccharide via anucleophile moiety or residue.

Many of the nitric oxide-nucleophile (NONOates) complexes have beenpromising as pharmacological compounds because, unlike,nitrovasodialators such as glyceryl trinitrite and sodium nitroprusside,they spontaneously release nitric oxide (NO) in aqueous solutions underphysiological conditions without first having to be metabolized.

Although the NONOate complexes release molecular nitric oxide (NO)without first having to be metabolized, pharmacological applicationshave been limited by their propensity to distribute evenly throughout agiven medium and their spontaneous release of nitric oxide (NO) inaqueous media, thus compromising site specific delivery of nitric oxide(NO) to target tissues.

Accordingly, there remains a great need to develop a low cost, readilybiodegradable, biocompatible nitric oxide donor polymer compositioncomprising a nitric oxide [N₂O₂]⁻ dimer and a medically beneficialcarrier molecule, capable of improved site specific delivery andcontrolled release of nitric oxide (NO) to target tissues underphysiological conditions, without the further side effects of the nitricoxide donor compounds disclosed in the prior art.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a chitosan-basedpolymeric nitric oxide donor composition comprising a modified chitosanpolymer and a nitric oxide [N₂O₂]⁻ dimer.

It is a further object of the present invention to providechitosan-based polymeric nitric oxide donor compositions in which thenitric oxide [N₂O₂]⁻ dimer is bound directly to the modified chitosanpolymer.

It is a further object of the present invention to provide achitosan-based polymeric nitric oxide donor composition which furtherincludes a medically beneficial carrier molecule.

It is a further object of the present invention to provide achitosan-based polymeric nitric oxide donor composition capable of sitespecific delivery and controlled release of nitric oxide underphysiological conditions.

It is a further object of the present invention to providechitosan-based polymeric nitric oxide donor compositions having firstorder nitric oxide release kinetics.

It is a further object of the present invention to provide a method ofpreparing a chitosan-based polymeric nitric oxide donor compositioncomprising reacting a nitric oxide (NO) dimer with a modified chitosanpolymer.

It is a further object of the present invention to provide achitosan-based polymeric composition to treat respiratory distress,emphysema, external wounds, internal wounds and for coating vasculargrafts.

These and other objects of the present invention, together with theadvantages over the prior art relating to nitric oxide donorcompositions, which should be become apparent from the specificationwhich follows, are accomplished by the composition and method which ishereafter described and claimed.

The present invention, therefore, provides a chitosan-based polymericnitric oxide donor composition comprising a modified chitosan polymerand a nitric oxide [N₂O₂]⁻ dimer, in which the nitric oxide [N₂O₂]⁻dimer is bound directly to a nitrogen atom in the backbone of themodified chitosan polymer. The chitosan-based nitric oxide donorcomposition of the present invention further comprises a medicallybeneficial carrier molecule. The chitosan-based nitric oxide donorcomposition has first order nitric oxide release kinetics and providessite specific delivery and controlled release of nitric oxide underphysiological conditions.

The nitric oxide donor composition of the present invention comprises achitosan polymer and a nitric oxide [N₂O₂ ⁻] dimer, said compositionhaving the general formula:

wherein R₁ is an organic group having from 1 to about 18 carbon atoms;

wherein x is from about 0.5 to about 0.8;

wherein y is from about 0.2 to about 0.5; and

wherein x and y are the mole fractions of each unit and the sum of x andy is 1.

The present invention further provides a method for preparing a nitricoxide donor composition comprising reacting a nitric oxide [N₂O₂]⁻ dimerwith a chitosan polymer, said chitosan polymer having the followinggeneral formula:

wherein R₁ is an organic group having from 1 to about 18 carbon atoms;

wherein x is from about 0.5 to about 0.8;

wherein y is from about 0.2 to about 0.5; and

wherein x and y are the mole fractions of each unit and the sum of x andy is 1.

In one preferred embodiment, the present invention further provides amethod for the preparing a nitric oxide donor composition comprisingreacting nitric oxide (80-100 p.s.i.) with a chitosan polymer in thepresence of sodium methoxide at room temperature, said chitosan polymerhaving the following general formula:

wherein R₁ is an organic group having from 1 to about 18 carbon atoms;

wherein x is from about 0.5 to about 0.8;

wherein y is from about 0.2 to about 0.5; and

wherein x and y are the mole fractions of each unit and the sum of x andy is 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the mechanism of synthesis andchemical structures of the chitosan-based nitric oxide donor compositionof the present invention.

FIG. 2 is a graph comparing the release profile of nitric oxide (NO)from the N-carboxybutyl chitosan NONOate (N—CBC—NO), N-carboxyethylchitosan NONate (N—CEC—NO) and N-caboxymethyl chitosan NONOate(N—CMC—NO) compositions of the present invention.

FIG. 3 is a graph comparing the release profile of nitric oxide (NO)from the N-propyl chitosan NONOate (N—PC—NO), N-carboxyethyl chitosanNONate (N—CEC—NO) and N-carboxymethyl chitosan methyl ester NONOate(N—CEC—Me ester-NO) compositions of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a chitosan-based polymeric compositioncapable of site specific delivery and controlled release of nitric oxideto target tissues comprising a modified chitosan polymer and a nitricoxide [N₂O₂]⁻ dimer. The nitric oxide (NO) dimer is bound or “loaded”onto the modified chitosan polymer. Thus, the modified chitosan polymeracts as a carrier of the nitric oxide (NO) dimer. The nitric oxide[N₂O₂]⁻ dimer is covalently bound to the modified chitosan polymer,forming a diazenium diolate (NONOate) derivative of the modifiedchitosan polymer. Chemical bonding of the nitric oxide [N₂O₂]⁻ dimer tothe modified chitosan polymer is achieved by covalent bonding of thenitric oxide [N₂O₂]⁻ dimer directly to a nitrogen atom in the backboneof the modified chitosan polymer, such that the nitric oxide [N₂O₂]⁻dimer becomes incorporated, or part of the modified chitosan polymer.The terminology modified chitosan polymer, as used throughout thespecification, is defined as any biologically compatible derivative ofchitosan.

It is known in the prior art to bond a nitric oxide-releasing [N₂O₂]⁻functional group or dimer to a polymer, such as a polysaccharide, via anucleophile moiety. In this situation, the nitric oxide (NO) dimer isbound to a nucleophile moiety forming a nitric oxide/nucleophilecomplex. The nitric oxide/nucleophile complex is subsequently chemicallyor physically bound to a polymer, such that the nitric oxide/nucleophilecomplex is associated with, becomes part of, incorporated with orcontained within the polymer matrix.

It has been discovered that a nitric oxide [N₂O₂]⁻ dimer can becovalently bonded to a nucleophilic nitrogen atom on the modifiedchitosan polymer, without first having to be bonded to a nuclephilicmoiety as described in the prior art. Specifically, the modifiedchitosan polymer is synthesized such that the primary amine in themodified chitosan polymer is converted into a secondary amine whichserves as the nucleophilic reaction site for the binding of the nitricoxide-releasing [N₂O₂]⁻ dimer.

The nitric oxide donor composition of the present invention comprises achitosan polymer and a nitric oxide [N₂O₂ ⁻] dimer, said compositionhaving the general formula:

wherein R₁ is an organic group having from about 1 to about 18 carbonatoms, x is from about 0.5 to about 0.8, y is from about 0.2 to about0.5 and x and y are the mole fractions of each unit and the sum of x andy is 1.

Preferably, the organic group, R₁, is an alkyl group having about 1 toabout 18 carbon atoms. The organic group may by straight or branchchained. The organic group may also contain heteroatoms, such asnitrogen, oxygen, sulfur and phosphorous. The organic group, R₁, maycontain an ester functionality, and may be represented by the followingformula:

wherein R₂ is an organic group having from about 1 to about 18 carbonatoms. R₂ may by straight or branch chained. R₂ may also containheteroatoms, such as nitrogen, oxygen, sulfur and phosphorous; andwherein R₃ is methyl. Preferably, R₂ is an alkyl group having from about1 to about 18 carbon atoms.

The organic group, R₁, may alternatively contain an acid functionality,and may be represented by the following formula:

wherein R₄ is an organic group having from about 1 to about 18 carbonatoms. R₄ may by straight or branch chained. R₄ may also containheteroatoms, such as nitrogen, oxygen, sulfur and phosphorous.Preferably, R₄ is an alkyl group having from about 1 to about 18 carbonatoms.

In one embodiment, the polymeric nitric oxide donor composition may alsocomprise a metal cation, M⁺, bonded to an oxygen atom on the nitricoxide dimer, to stabilize the charge on the polymer. In this embodiment,the polymeric nitric oxide donor may be represented by the followinggeneral formula:

Without limitation, the metal cation, M⁺, may be selected from cationsof alkali metals. Preferably, the metal cation is Na⁺.

Alternatively, the polymeric nitric oxide donor composition may containpositive charge on amine functionality of the chitosan polymer tostabilize the charge on the polymer. In this embodiment, the polymericnitric oxide donor may be represented by the following general formula:

The present invention provides a chitosan-based NONOate compositionwherein the composition further comprises a medically beneficialcarrier. It is known that modified chitosan polymers, such asN-carboxybutyl chitosan, possess medically beneficial properties. Forexample, wound dressings incorporating N-carboxybutyl chitosan stimulateordered regeneration and vascularization of tissue and allow for gasexchange, resulting in a reduction of secondary infections and scarformation. (Muzzarelli, R. et al., “N-carboxybutyl Chitosan as a WoundDressing and a Cosmetic Ingredient”, Chim. Oggi. Vol 9, No. 4, 33-37(1991); Biagini, Graziella et al. “Wound Management with N-carboxybutylChitosan”, Biomaterials, Vol. 12, 281-286 (1991); Biagini, Graziella etal. “Morphological Study of the Capsular Organization Around TissueExpanders Coated with N-carboxybutyl Chitosan”, Biomaterials, Vol. 12,287-291 (1991). It is also known that N-carboxybutyl chitosan possessessignificant antibacterial properties against candidae, staphylococci,streptococci and enterococci. (Muzzarelli, R. et al. “AntimicrobialProperties of N-carboxybutyl Chitosan.” Antimicrobial Agents andChemotherapy, 2019-2023 (1990).

By way of exemplification, but not in limitation, suitable modifiedchitosan polymers include N-carboxymethyl chitosan (N—CMC),N-carboxyethyl chitosan (N—CEC), the methyl ester of N-carboxyethylchitosan (N—CEC—Me ester), N-carboxybutyl chitosan (N—CBC), N-propylchitosan (N—PC), and the like. The modified chitosan polymers employedin the present invention have the following general chemical formula:

wherein R₁ is an organic group having from 1 to about 18 carbon atoms.R₁ may also contain heteroatoms, such as nitrogen, oxygen, sulfur andphosphorous. R₁ may also contain ester or acid functionalities.Preferably, the organic group, R₁, is a straight or branched chain alkylgroup having from about 1 to about 18 carbon atoms;

x is from about 0.5 to about 0.8;

y is from about 0.2 to about 0.5; and

wherein x and y represent the mole fraction of each unit and the sum ofx and y is 1.

More preferably, the organic group, R₁, is selected fromCH₃CHCH₂CH₂COO⁻, CH₂CH₂COO⁻, CH₂CH₂COOCH₃, CH₂COO⁻ and CH₂CH₂CH₃.

Synthesis of N-Carboxybutyl Chitosan (N—CBC) and N-CarboxymethylChitosan (N—CMC)

The modified chitosan polymers, N-carboxybutyl chitosan (N—CBC) andN-carboxymethyl chitosan (N—CMC), employed in the present invention weresynthesized by the method of Muzzarelli, U.S. Pat. No. 4,835,265,incorporated herein by reference. N-carboxybutyl chitosan (N—CBC) hasthe following chemical formula:

and N-carboxymethyl chitosan (N—CMC) has the following chemical formula:

wherein:

x is from about 0.5 to about 0.8;

y is from about 0.2 to about 0.5; and

wherein x and y represent the mole fraction of each unit and the sum ofx and y is 1.

Briefly, 3.6 grams of chitosan was dissolved in 80 milliliters of H₂O,containing 50% weight percent levulinic acid and glyoxilic acid (1.5times the molar amount of chitosan). The addition of levulinic acid andglyoxilic acid to the chitosan results in the formation of a Schiff'sbase. Twelve hours after the reduction of the Schiff's bases, the pH ofthe was adjusted to 7.0 with 0. 1M NaOH. N-carboxybutyl chitosan (N—CBC)was subsequently dialyzed against distilled H₂O for three days andfreeze dried to obtain a soft spongy product having a percent yield of58 percent. N-carboxymethyl chitosan precipitated at 7.0, and wasfiltered off and washed in an ethanol solution. The N-carboxymethylchitosan (N—CMC) was freeze dried and had a percent yield of 87 percent.

Synthesis of N-Carboxyethyl Chitosan (N—CEC) and the Methyl Ester ofN-Carboxyethyl Chitosan (N—CEC—Me Ester)

N-carboxyethyl chitosan (N—CEC) and the methyl ester of N-carboxyethylchitosan (N—CEC—Me ester) were synthesized by the method of Bartowiak etal, incorporated herein by reference. N-carboxyethyl chitosan (N—CEC)has the following chemical formula:

and the methyl ester of N-carboxyethyl chitosan (N—CEC—Me ester) has thefollowing formula:

wherein:

x is from about 0.5 to about 0.8;

y is from about 0.2 to about 0.5; and

wherein x and y represent the mole fraction of each unit and the sum ofx and y is 1.

Briefly, 2.0 grams of chitosan dissolved in 100 milliliters of 0.1 molaracetic acid was reacted with 4.5 milliliters of methyl acrylate (5 timesthe molar amount of chitosan) in 70 milliliters of methanol (MeOH) via aMichael addition reaction. The reaction was stirred at room temperaturefor 48 hours, resulting in a viscous product. The viscous product wasprecipitated in an mixture of 400 milliliters of methanol (MeOH) and 250milliliters of ammonium hydroxide (NH₄OH) and subsequently filtered.After soxhlet extraction with methanol (MeOH), 2.25 grams of methylester of N-carboxyethyl chitosan (N—CEC) was obtained. N-carboxyethylchitosan (N—CEC) was obtained by treating the methyl ester with aMeOH-aqueous NaOH solution for 3 hours at 60° C.

Synthesis of N-Propyl Chitosan (N—PC)

N-propyl chitosan (N—PC) was synthesized by the method of Muzzarelli etal, U.S. Pat. No. 4,835,265, incorporated herein by reference. N-propylchitosan (N—PC) has the following chemical formula:

wherein:

x is from about 0.5 to about 0.8;

y is from about 0.2 to about 0.5; and

wherein x and y represent the mole fraction of each unit and the sum ofx and y is 1.

Briefly, 2 grams of chitosan dissolved in 1% acetic acid was reactedwith 1.36 milliliters of an aqueous solution of propionaldehyde (2 timesthe molar amount of chitosan). After 30 minutes, 0.1 molar NaOH wasadded to the chitosan solution to increase the pH to 4.5, forming aSchiff s base. The Schiff's base was reduced by the addition of 0.5grams of NaBH₄ in 5 milliliters of H2O, and stirring for 12 hours. ThepH was susequently adjusted to 10 to precipitate the N-propyl chitosan(N—PC). Soxhlet extraction was performed for 24 hours with ethanol(EtOH), and another 24 hours with diethylether was performed to removethe remaining aldehyde and inorganic products. The percent yieldobtained for N-propyl chitosan (N—PC) was 77 percent.

Synthesis of Chitosan-based NONOate Compositions

The chitosan-based diazenium diolates (NONOates) of the presentinvention were synthesized by reacting nitric oxide gas with themodified chitosan polymers described hereinabove. The above modifiedchitosan polymers were synthesized so that the primary amine in thechitosan polymer was converted into a secondary amine that serves as thenucleophile reaction site for the binding of the nitric oxide (NO)dimer. Approximately 0.5 grams of the chitosan derivatives,N-carboxybutyl chitosan (N—CBC), N-carboxymethyl chitosan (N—CMC),N-carboxyethyl chitosan (N—CEC), methyl ester of N-carboxyethyl chitosan(N—CEC—Me ester) and N-propyl chitosan (N—PC), were suspended in 40milliliters of sodium methoxide in methanol solution, and placed in hightemperature glass (Ace Glass) bottles equipped with a magnetic stir bar.The solutions were exposed to 100 psi of fresh nitric oxide gas for onehour per day for 7 consecutive days. The resulting products werefiltered off. The isolated products were stored in air tight containersat −20° C. in a dessicator. Medium molecular weight chitosan(190,000-300,000) with a deacetylation degree between 75%-85%, levulinicacid, glyoxilic acid, methyl acrylate, propionaldehyde and sodiumcyanoborohydride were purchases from Aldrich Chemical Co., and were usedwithout further purification. Nitric oxide gas was purchased fromMatheson Gas Products, Inc. All other materials were reagent grade andwere purchased from Fisher Scientific, Inc.

Release Profile of Chitosan-based NONOate Compositions

NONOates release NO in aqueous media, and the rate at which NO isreleased is dependant on the pH and temperature of the media. Therelease profile for the various chitosan-based NONOate compositions ofthe present invention was measured using a nitric oxide (NO) analyzer.The nitric oxide analyzer was connected to a 150 milliliter samplechamber consisting of a gas impinger bottle modified with two-way valvesthat allowed the nitric oxide (NO) gas to accumulate in the samplechamber. The 150 milliliter sample chamber was filled with 25milliliters of Phosphate Buffered Saline (PBS; pH=7.4), and the solutionwas degassed for 15 minutes with helium (10 psig at 150 mil/minute). Therelease profile for each of the chitosan-based NONOate compositions ofthe present invention was individually determined. Five (5) milligramsamples of the chitosan-based NONOate compositions of the presentinvention were added to the sample chamber, the valves were closed andperiodic readings were taken by opening the valves and allowing thenitric oxide released from the chitosan-based NONOate compositions ofthe present invention to be swept into the detector by way of the heliumgas.

The release profiles of nitric oxide (NO) from N-carboxybutyl chitosanNONOate (CBC—NO), N-carboxyethyl chitosan NONOate (CEC—NO) andN-carboxymethyl chitosan NONOate (CMC—NO) are shown in FIG. 2. Therelease profile data for N-carboxybutyl chitosan NONOate (CBC—NO)contained in FIG. 2, demonstrates that about 250 nanomoles of nitricoxide (NO) is released in an aqueous medium at 37° C. within 40 hoursafter the N-carboxybutyl chitosan NONOate (CBC—NO) of the presentinvention was placed in the aqueous medium. The release profile data forN-carboxyethyl chitosan NONOate (CEC—NO) contained in FIG. 2,demonstrates that about 150 nanomoles of nitric oxide (NO) is releasedin an aqueous medium at 37° C. within 40 hours after adding theN-carboxyethyl chitosan NONOate (CEC—NO) composition of the presentinvention in the aqueous medium. The release profile data forN-carboxymethyl chitosan NONOate (CMC—NO) contained in FIG. 2,demonstrates that about 35 nanomoles of nitric oxide (NO) is released inan aqueous medium at 37° C. within 40 hours after the N-carboxymethylchitosan NONOate (CMC—NO) of the present invention added to the aqueousmedium.

The release profiles of nitric oxide (NO) from N-propyl chitosan NONOate(N—PC—NO), N-carboxyethyl chitosan NONOate (N—CEC—NO) and the methylester of N- carboxyethyl chitosan NONOate (CEC—Me ester—NO) was shown inFIG. 3. The release profile data for N-propyl chitosan NONOate (N—PC—NO)contained in FIG. 3, demonstrates that about 85 nanomoles of nitricoxide (NO) is released into an aqueous medium at 37° C. within 40 hoursafter adding the N-propyl chitosan NONOate (N—PC—NO) composition of thepresent invention to the aqueous medium. The release profile data forN-carboxyethyl chitosan NONOate (N—CEC—NO) shown in FIG. 3, againdemonstrates that about 150 nanomoles of nitric oxide (NO) is releasedfrom N-carboxyethyl chitosan NONOate (N—CEC—NO) in an aqueous medium at37° C. within 40 hours after adding the N-carboxyethyl chitosan NONOate(N—CEC—NO) of the present invention to the aqueous medium. The releaseprofile data for the methyl ester of N-carboxyethyl chitosan NONOate(CEC—Me-ester-NO) shown in FIG. 3, demonstrates that about 75 nanomolesof nitric oxide (NO) is released from the methyl ester of N-carboxyethylchitosan NONOate (CEC—Me-ester-NO) of the present invention in anaqueous medium at 37° C. within 40 hours after adding the methyl esterof N-carboxyethyl chitosan NONOate (CEC—Me-ester-NO) to the aqueousmedium.

Release kinetic measurements were determined by calculating theconcentration of nitric oxide (NO) released from carboxybutyl chitosanNONOate (CBC—NO) with a KNO₃ standard curve (100 μmol/L). A releaseprofile was obtained by plotting the cumulative sum of nitric oxide (NO)produced (nanomoles) versus time (hours). From the graph of the releaseprofile, the concentration of nitric oxide (NO) at infinity wasdetermined. The first order rate was calculated by plotting:ln([NO]_(∞)−[NO]_(t)) versus time. From this calculation, the k valueand the half of carboxybutyl chitosan NONOate (CBC—NO) was determined.The half lives (t½) of chitosan-based NONOate compositions of thepresent invention are shown in Table I below.

TABLE I COMPOUND HALF LIFE (T_(1/2)) OF NO RELEASE N-CBC-NO  244 min.N-CEC-NO  90 min. N-CEC Me ester-NO 198 min. N-CMC-NO  13 min. N-PC-NO300 min.

Thus, it is demonstrated by FIGS. 2 and 3, and Table I above, that thechitosan-based NONOate compositions of the present invention are capableof controlled release of nitric oxide (NO) in aqueous media. It isfurther demonstrated that the optimal temperature and pH for release ofnitric oxide (NO) from the chitosan-based NONOate compositions of thepresent invention is 37° C. and 7.4, respectively.

The chitosan-based NONOate compositions of the present invention have awide variety of medical applications in which an effective dosage ofnitric oxide is indicated as a preferred treatment. By way ofillustration, and not in limitation, the chitosan-based NONOatecompositions of the present can be used in the following medicalapplications for treatment of conditions or disorders in which aneffective dosage of nitric oxide is indicated.

It is known that the vascular endothelium produces nitric oxide (NO)which acts as a vasodilator of blood vessels. Many cardiovascular andpulmonary disorders, such as pulmonary hypertension, myocardial ischemiaand reperfusion, atherosclerosis and congestive heart failure, are aresult of inability of the vascular endothelium to producephysiologically significant levels of nitric oxide (NO). Pulmonaryhypertension, for example, results from a block of nitric oxide (NO)synthesis. The chitosan-based NONOate compositions of the presentinvention can be incorporated into dry powder inhalers, nose drops andaerosol formulations to be administered via inhalation for the sitespecific delivery and controlled release of nitric oxide (NO) to thepulmonary vasculature; the release nitric oxide (NO) having avasodilatory effect on the blood vessels, with a corresponding decreasein systolic blood pressure.

It is known that nitric oxide synthesis by macrophages, neutrophils andother inflammatory cells are increased during inflammation and tissueremodeling in normal wounds. It is also known that chitosan derivatives,such as N-carboxybutyl chitosan have significant wound healing andantimicrobial effects. N-carboxybutyl chitosan promotes orderedconnective tissue regeneration and decreases in scar formation and woundcontraction. The gel forming ability, ease of sterilization and absenceof side reaction products of N-carboxybutyl chitosan make it anattractive choice as a wound dressing. Therefore, the chitosan-basedNONOate compositions of the present invention has the benefit of nitricoxide (NO) delivery and further medically beneficial properties of themodified chitosan polymer, and may be incorporated into gels, creams andointments for the topical delivery of nitric oxide (NO) to dermalwounds, especially in patients with compromised macrophage function.

It is also known that endothelium derived nitric oxide (NO) inhibitsplatelet aggregation and adhesion when platelets come into contact withwalls of blood vessels. The chitosan-based NONOate composition of thepresent invention may be coated onto stents and implants useful forinhibition of platelet aggregation and adhesion to blood vessel wallsfollowing medical procedures, such as angioplasty.

Male erection involves neuronally mediated vasorelaxation of the bloodvessels of the smooth muscle of the corpora cavernosa. Although theexact mechanism is not fully elucidated, it is known that nitric oxide(NO) mediates male penile erection through relaxation the smooth muscleof the corpora cavernosa. Several in vivo studies have demonstrated thatnitric oxide (NO) is the principal neurotransmitter in cavernous smoothmuscle. Wang, R., et al., Nitric Oxide mediates penile erection incats,” The Journal of Urology 1994, Vol. 151:234-237. Therefore, thechitosan-based NONOate compositions of the present invention mayincorporated into topical hydrophilic gels, creams and lubricants,sprays, aerosols, penile implants, dermal patches and condoms, fortreatment of penile erection dysfunction in males.

The chitosan-based NONOate compositions of the present invention areuseful in any situation when it is desirable to deliver an effectivedosage of nitric oxide. Depending on the needs of the particularpatient, the chitosan-based NONOate composition of the present inventionmay be incorporated into dry powder inhalers, aerosols or nose drops tobe administered via the nasopharynx. In addition, the chitosan-basedNONOate composition of the present invention may be administered by adevice inserted into the oropharynx or naseopharynx, or via anendotracheal tube in patients with a tracheostomy.

One skilled in the art will recognized that there various methods ofadministration of the chitosan-based NONOate composition of the presentinvention, including, but not limited to, oral administration,inhalation administration, topical administration and parenteraladministration. The following methods of administration are provided asillustration, but not of limitation, of the preferred methods ofadministration of the chitosan-based NONOate composition of the presentinvention.

The chitosan-based NONOate composition of the present invention can beincorporated into formulations suitable for oral administration.Formulations suitable for oral administration include liquid solutions,tablets, capsules, lozenges, suspensions and emulsions.

The chitosan-based NONOate composition of the present invention can beincorporated into dry powder inhalers, nose drops and aerosolformulations to be administered via inhalation. The aerosol formulationscan be placed into acceptable pressurized propellants, such asdichlorofluoromethane, propane, nitrogen, and the like. Although morethan one method of administration may be employed, a particular methodof administration can be determined based on the specific needs of thepatient.

The chitosan-based NONOate composition of the present invention can beincorporated into formulations suitable for parenteral or intravenousadministration. Suitable formulations for parenteral or intravenousadministration include aqueous and non-aqueous, isotonic, sterileinjection solution which may further contain antioxidants, buffers,preservatives, solubilizing agents, chelating agents, stabilizers andthickening agents.

Thus, it is demonstrated that the objects of the present invention aremet. The examples included above are for illustrative purposes only andthe chitosan-based NONOate composition of the present invention are notlimited to them. It is to be understood that the chitosan-based NONOatecompositions of the present invention may be incorporated into variousdelivery systems, and thus, the selection of specific mode of deliverycan be determined based on the needs of the patient, without departingfrom the spirit of the invention herein disclosed and described. Thus,the scope of the invention shall include all modifications andvariations that may fall within the scope of the attached claims andequivalent embodiments.

We claim:
 1. A nitric oxide donor composition containing a chitosanpolymer having nitric oxide (N₂O₂—) groups bonded thereto, saidcomposition having the general formula:

wherein R₁ is an organic group having from 5 to about 18 carbon atoms;wherein x is from about 0.5 to about 0.8; wherein y is from about 0.2 toabout 0.5; and wherein x and y are the mole fractions of each unit andthe sum of x and y is
 1. 2. The nitric oxide donor composition of claim1, wherein R₁ is selected from N-propyl, N-carboxymethyl,N-carboxyethyl, N-carboxybutyl and the methyl ester of N-carboxyethyl.3. The nitric oxide donor composition of claim 2, wherein R₁ isN-carboxybutyl.
 4. The nitric oxide donor composition of claim 1,wherein said nitric oxide donor composition is capable of site specificdelivery and controlled release of nitric oxide under physiologicalconditions.
 5. The nitric oxide donor composition of claim 1, whereinsaid nitric oxide donor composition exhibits first order nitric oxiderelease kinetics.
 6. The nitric oxide donor composition of claim 1,wherein said nitric oxide donor composition contained a nitric oxidecarrier molecule possessing medically beneficial properties.
 7. Thenitric oxide donor composition of claim 1, wherein said nitric oxidedonor composition is useful for the treatment of respiratory distress,emphysema, external wounds, internal wounds and coating vascular grafts.8. A gel comprising the nitric oxide donor composition of claim
 1. 9. Acream comprising the nitric oxide donor composition of claim
 1. 10. Anointment comprising the nitric oxide donor composition of claim
 1. 11. Adermal patch comprising the nitric oxide donor composition of claim 1.12. A wound dressing comprising the nitric oxide donor composition ofclaim
 1. 13. A medical stent coated with the nitric oxide donorcomposition of claim
 1. 14. An medical implant co ated wi th the nitricoxide donor co mposition of claim
 1. 15. A condom coated with the nitricoxide donor composition of claim
 1. 16. A dry powder inhaler comprisingthe nitric oxide donor composition of claim
 1. 17. A nose dropcomprising the nitric oxide donor composition of claim
 1. 18. An aerosolspray comprising the nitric oxide donor composition of claim
 1. 19. Atablet comprising the nitric oxide donor composition of claim
 1. 20. Acapsule comprising the nitric oxide donor composition of claim
 1. 21. Alozenge comprising the nitric oxide donor composition of claim
 1. 22. Asuspension comprising the nitric oxide donor composition of claim
 1. 23.An emulsion comprising the nitric oxide donor composition of claim 1.24. A parenteral formulation comprising the nitric oxide donorcomposition of claim
 1. 25. A method for preparing a nitric oxide donorcomposition comprising reacting nitric oxide with a chitosan polymer,said chitosan polymer having the following general formula:

wherein R₁ is an organic group having from 5 to about 18 carbon atoms;wherein x is from about 0.5 to about 0.8; wherein y is from about 0.2 toabout 0.5; and wherein x and y are the mole fractions of each unit andthe sum of x and y is
 1. 26. The method of claim 25, wherein R₁ isselected from N-propyl, N-carboxymethyl, N-carboxyethyl, N-carboxybutyland the methyl ester of N-carboxyethyl.
 27. The method of claim 26,wherein R₁ is N-carboxybutyl.
 28. The method of claim 25, wherein themethod comprises reacting a nitric oxide dimer at a pressure of 80-100p.s.i. with said chitosan polymer in the presence of sodium methoxide atroom temperature.